Previous research has shown that inactivation of the tRNA 2-thiouridine synthesizing protein (tus) pathway leads to impaired bacteriophage lambda infection, while inactivation of the iron-sulfur clusters (isc) pathway facilitates lambda infection relative to wild-type E. coli. The overarching objective of the proposed research is to understand how these two pathways, which share a common node at cysteine desulferase IscS, affect lambda replication in its host-E. coli. IscS transfers sulfur down these pathways through direct binding of IscU and TusA. The tus pathway is required for 2-thiolation of U34 (s2U34) in tRNAGlu, tRNAGln, and tRNALys. This modification is conserved in all branches of life and has been shown to improve translation dynamics and reduce unwanted frameshifting. Interestingly, in lambda phage the product ratio of tail region proteins gpG and gpGT is controlled by ribosomal frameshifting. A specific "slippery sequence" encoding the dipeptide -Gly-Lys- slips in the -1 direction about 3.5% of the time, creating the longer fusion protein gpGT. While not part of the final tail structure, the precise gpG-gpGT ratio is necessary for proper lambda tail formation. I hypothesize that when sulfur relay down the tus pathway is disrupted, the hypomodified tRNALys increases the frequency of frameshifting at the "slippery sequence" between gpG and gpGT, affecting lambda phage tail synthesis. I will investigate this by (1) examining potential epistatic effects between tusA and iscU on lambda replication, (2) determining the effect of s2U34 on the gpG to gpGT ratio and the resulting viable phage, and (3) determining if impaired lambda phage replication in mutants for s2U34 is, in part, a result of general translational defects. This research should elucidate of the interplay between the isc and tus pathways in lambda phage infection and should be relevant to other tailed bacteriophages and mammalian viruses (like HIV) which use "slippery sequences" to control gene expression ratios. In the end, this work should lead to a deeper understanding of viral replication and innovative antiviral strategies at the translational level. PUBLIC HEALTH RELEVANCE: The overarching objective of the proposed research is to investigate a new technique for disrupting virus replication. My research is potentially applicable to all viruses that use a technique called programmed frameshifting to control its protein ratios. I am hopeful that this work will have a dramatic impact on our understanding of virus replication and could lead to new antiviral strategies. !